PLA2 inhibitory compounds

ABSTRACT

The present invention provides peptides and compounds which inhibit the enzyme activity of Type II phospholipases A 2 . The preferred compounds are pentapeptides. Where the phospholipase is human Type II phospholipase A 2  the preferred peptides are FLSYK and KFLSY.

FIELD OF THE INVENTION

The present invention relates to peptides which inhibit the enzymaticactivity of phospholipases A₂ (PLA₂ s) and illustrated with peptideswhich inhibit the activity of Type II PLA₂ 's particularly synovial PLA₂and snake PLA₂ (Crotalus durissus and Crotalus atrox). In addition, thepresent invention relates to pharmaceutical composition including, asthe active ingredient these peptides and to methods of treatmentinvolving the administration of this composition.

BACKGROUND OF THE INVENTION

Phospholipases A₂ constitute a diverse family of enzymes with twosubclasses (Type I and Type II) (FIG. 1), based on the positions of thedisulphide bonds in the molecules while bee venom PLA₂ constitutes athird substantially distinct class of PLA₂. X-ray crystallography hasrevealed that the segments comprising the functional substructure of theenzyme is similar in classes. This similarity is particularly strikingwhen the structurally-related Type I/II enzymes are compared with beevenom enzyme (2). PLA₂ hydrolyses the sn-2 acyl ester bond ofphosphoglycerides initiating the release of fatty acid precursors ofinflammatory eicosanoids. Human synovial PLA₂ (a Type II molecule) hasrecently been isolated and identified (3). The same PLA 2 has beenimplicated in the pathogensis of several inflammatory diseases in humanssuch as rheumatoid arthritis and Gram negative septic shock (7;8).

Murine, inhibitory monoclonal antibodies raised against synovial PLA₂have demonstrated pre-clinical efficacy. Accordingly, there is interestin the development of compositions which inhibit the enzymatic activityof PLA₂.

Tryptic digestion of human synovial PLA₂ and subsequent separation andanalysis of the fragments by EPLC gave a very interesting and unexpectedresult for one of the peaks in that it contained two peptides; one aheptapeptide (the N-terminal peptide) and the other a pentapeptide,FLSYK (SEQ ID NO:8) (corresponding to residues 70-74 in other PLA₂molecules, based on three-dimensional structural "homology" of mammalianPLA₂ amino acid sequences (1,4)). The pentapeptide was found in anearlier eluting, fully resolved peak (as expected). Since the HPLCsystem failed to fully resolve these two peptides in the latter peak,these data suggest that the two peptides had a strong affinity for oneanother and raised questions as to the structural implications of this.X-ray diffraction studies (5,6) have shown that amino acid residues inthe two peptides are close to the active site of the enzyme and areimportant in forming or stablising the channel in which the1,2-diacyl-3-sn-phosphoglyceride substrate is precisely positioned forhydrolysis of the 2-ester bond. The first turn of the N-terminal helix(residues 1 to 12) is stablised by a hydrogen bond network provided bythe N-terminus and residue 4, elements of residues 69 to 71 and a watermediated link to the catalytic residues; residues 2 and 5 form the"floor" of the channel, residue 9 forms the right wall and the left wallis formed by residue 69 (either tyrosine or lysine usually) which ispredicted to move after the substrate has docked and to form a hydrogenbond with the sn-3 phosphate of the substrate. The chemical evidence ofthe strong interactions between the heptapeptide and the pentapeptideprompted the supposition that the PLA₂ activity may be inhibited in thepresence of either one of these peptides.

Using synthetic peptide chemistry the present inventors have preparedthe pentapeptide FLSYK and demonstrated that addition of it to the assaymedium decreased the enzyme activity of human synovial PLA₂ (FIG. 2a).Furthermore, it has been demonstrated that the pentapeptide thatoccupies the 70-74 region of snake PLA₂ (WDIYR) also inhibited theactivity of snake PLA₂ (see FIG. 3b). It is believed that thisinhibition is mediated by the peptide binding to the amino terminal endof the enzyme and blocking the reaction either by blocking the substrateaccess to the hydrophobic channel or by distorting the structuresufficiently to prevent correct orientation of the substrate.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the present invention consists in alinear or cyclic peptide of at least 5 residues which inhibits theenzymatic activity of human synovial PLA₂, the peptide having thefollowing formula:

    A.sub.1 -A.sub.2 -A.sub.3 -A.sub.4 -A.sub.5 -A.sub.6 -A.sub.7

in which

A₁ is hydrogen or one or two naturally occurring amino acids

A₂ is F or Y or W or absent

A₃ is L or V or I or M

A₄ is S or T

A₅ is Y or F or W

A₆ is K or R or H or absent

A₇ is OH or one or two naturally occurring amino acids.

In a preferred embodiment the peptide is a pentapeptide.

In another preferred embodiment of the present invention A₁ is H and A₇is OH.

In a further preferred embodiment of the present invention the peptideis FLSYK (SEQ ID NO:8) or KFLSY (SEQ ID NO:9) and most preferably FLSYK.

In a second aspect the present invention consists in a linear or cyclicpeptide of at least 5 residues which inhibits the enzymatic activity ofCrotalus durissus PLA₂, the peptide having the following formula:

    B.sub.1 -B.sub.2 -B.sub.3 -B.sub.4 -B.sub.5 -B.sub.6 -B.sub.7

in which

B₁ is hydrogen or one or two naturally occurring amino acids

B₂ is W or F or Y or absent

B₃ is D or E

B₄ is I or V or L or M

B₅ is Y or F or W

B₆ is R or K or H or absent

B₇ is OH or one or two naturally occurring amino acids.

In a preferred embodiment the peptide is a pentapeptide.

In another preferred embodiment of the present invention B₁ is H and B₇is OH.

In a further preferred embodiment of the present invention the peptideis WDIYR (SEQ ID NO:10).

In a third aspect the present invention consists in a linear or cyclicpeptide of at least 5 residues which inhibits the enzymatic activity ofCrotalus atrox PLA₂, the peptide having the following formula:

    C.sub.1 -C.sub.2 -C.sub.3 -C.sub.4 -C.sub.5 -C.sub.6 -C.sub.7

in which

C₁ is hydrogen or one or two naturally occurring amino acids

C₂ is T or S or absent

C₃ is V or I or L or M

C₄ is S or T

C₅ is Y or F or W

C₆ is T or S or absent

C₇ is OH or one or two naturally occurring amino acids.

In a preferred embodiment the peptide is a pentapeptide.

In another preferred embodiment of this aspect of the present inventionC₁ is H and C₇ is OH.

In a further preferred embodiment of this aspect of the presentinvention the peptide is TVSYT (SEQ ID NO:11).

As will be clear to those skilled in the art from the disclosureprovided herein, the peptides of the first and second aspect of thepresent invention illustrate how the enzymatic activity of other PLA₂ smay be inhibited. This inhibition may be achieved by compounds whichinteract with the N-terminal amino acid sequence of the PLA₂ molecule ina manner such that the channel into which the phospholipid diffusesprior to catalytic cleavage is destabilized.

Accordingly, in a fourth aspect the present invention consists in acompound which inhibits the enzymatic activity of phospholipase A₂, thecompound being characterized in that it interacts with the N-terminalamino acid sequence of the phospholipase A₂ such that the channel intowhich the phospholipid diffuses prior to catalytic cleavage is eitherblocked or destabilized.

In a preferred embodiment of the present invention the PLA₂ is humanPLA₂ and the compound is a peptide.

In a preferred embodiment of the present invention the peptide has theamino acid sequence FLSYK or KFLSY.

As will be clear to those skilled in the art, the present inventors havefound that the enzymatic activity of a phospholipase A₂ can be inhibitedby a peptide having a sequence corresponding to a sequence selected fromthe region of residues 69 to 75 of the phospholipase 2.

Accordingly, in a fifth aspect the present invention consists in apeptide of 5 or 6 residues which inhibits the enzymatic activity of aphospholipase A₂, the peptide having an amino acid sequencecorresponding to a sequence selected from the region of residues 69-75of the phospholipase A₂.

In a preferred embodiment this aspect of the present invention thepeptide is a pentapeptide and has an amino acid sequence correspondingto the sequence from residue 69-73 or 70-74 of the phospholipase A₂.

In a further preferred embodiment of the present invention thephospholipase A₂ is human phospholipase A₂.

In a sixth aspect the present invention consists in a composition foruse in treating a subject suffering from septic shock rheumatoidarthritis and/or other inflammatory diseases, the composition comprisinga therapeutically acceptable amount of peptide or compound of the first,fourth or fifth aspect of the present invention and a pharmaceuticalacceptable sterile carrier.

In a seventh aspect the present invention consists in a method oftreating septic shock and/or inflammatory disease in a subjectcomprising administering to the subject the composition of the sixthaspect of the present invention.

It will be appreciated by those skilled in the art that a number ofmodifications may be made to the peptides of the present inventionwithout deleteriously effecting the biological activity of the peptide.This may be achieved by various changes, such as insertions, deletionsand substitutions, either conservative or non-conservative in thepeptide sequence where such changes do not substantially decrease thebiological activity of the peptide. By conservative substitutions theintended combinations are:

G, A; V, I, L, M; D, E; N, Q; S, T; K, R, H; and F, Y, W.

It may also be possible to add various groups to the peptide of thepresent invention to confer advantages such as increased potency orextended half life in vivo, without substantially decreasing thebiological activity of the peptide.

It is intended that such modifications to the peptide of the presentinvention which do not result in a decrease in biological activity arewith in the scope of the present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following examples and Figures, in which:

FIG. 1 shows mammalian PLA₂ amino acid sequences (SEQ ID NOS. 1, 2, 3,4, 5, 6 and 7).

FIG. 2: Inhibition of human PLA₂ using the peptide FLSYK.

FIG. 2(a) was obtained using a peptide from a tryptic digest of theenzyme (n=7 control ♦ inhibitor), 2(b) and 2(c) with a synthetic peptiden=11 control ♦ inhibitor control ♦ inhibitor, respectively. Thesynthetic peptide also inhibits the enzyme in septic shock serum [FIG.2(c)].

FIG. 3: Dose response curves showing increasing inhibitor withincreasing amount of FLSYK and human recombinant Type II PLA₂ (3ainhibitor ♦ control) and in PLA₂ in septic shock serum (3b inhibitor ♦control).

FIG. 4: Dose response curves for FLSYK (4a PLA₂ ♦ control) and WDIYR (4bsnake (II) ♦ control) on human PLA₂ and snake (Crotalus Durissus) PLA₂respectively. Both peptides occupy similar sites in their parentproteins and show inhibitory properties for the enzymatic activity.

FIG. 5 shows a Lineweaver-Buspe plot showing inhibition of PLA₂ by FLSYK(PLA₂ ♦ 10 ug ▪ FLSYK, ⋄ 1 ug FLSYK).

INHIBITION OF PLA2 ACTIVITY

Proteins and Peptides

1. Synovial PLA₂, snake PLA₂ (Crotalus Durissus and Crotalus ATR?)

2. Phe-Leu-Ser-Tyr-Lys (FLSYK) (SEQ ID NO:8)

3. Acetyl-Phe-Leu-Scr-Tyr-Lys-Methyl ester (Ac-FLSYK-OMe)

4. Trp-Asp-Ile-Tyr-Arg (WDIYR) (SEQ ID NO:10)

5. Lys-Phe-Leu-Ser-Tyr (KFLSY) (SEQ ID NO:9)

6. Thr-Val-Ser-Tyr-Thr (TVSYT) (SEQ ID NO:12)

7. Phe-Lys-Thr-Tyr-Ser (FKTYS) (SEQ ID NO:13)

8. Thr-Glu-Ser-Tyr-Ser (TESYS) (SEQ ID NO:14)

9. Gly-Thr-Lys-Phe-Leu-Ser-Tyr-Lys-Phe-Ser-Asn (GTKFLSYKFSN) (SEQ IDNO:15)

10. Lys-Phe-Leu-Ser-Tyr-Tyr (KFLSYY) (SEQ ID NO:16)

11. Phe-Leu-Ser-Tyr (FLSY) (SEQ ID NO:17)

12. Phe-Leu-Ser-Tyr-Lys-NH₂. (FLSYK-NH₂)

Tryptic Digestion of PLA2:

Approximately 100 μg of PLA 2 was dissolved in 300 μl of 1 MTris pH 8.015 μl of Trypsin solution (10μ/1M Tris pH 8) was added and thepeptide/trypsin solution was incubated for 2 hours at 37° C. 5 μl ofneat TFA was used to lower the. pH to terminate the digestion. The wholesolution was subjected to microbore HPLC fractionation.

Microbore HPLC fractionation:

An ABI Microbore syringe pump system Model 140 was used. Detectorwavelength was set at 220 nm at 0.5 aufs. A RP-300 1×100 mm was used.Fractionation was carried out by running a linear buffer gradient from0.1% TFA in water to 0.1% TFA, 70% acetonitrile in water over sixtyminutes. Amino acid sequences identified from fractions were:

Fraction #2 (K)YQYYSNK

Fraction #4 FLSYK

Fraction #5 FLSYK NLVNFHR

Fraction #7* EALLSYGFYG(C)H(C)GVGGR (C)(C)VTHD(C)(C)YK SQL(C)E(C)DKIT(C)AK AAAT(C)FAR

Fraction #9 EAALSYGFYG

Peptide Synthesis:

Peptide synthesis was carried out in an ABI Peptide Synthesiser Model430A. T-Boc chemistry was used. HF cleavage was used to release peptidefrom the solid support.

PLA2 Serial Dilution:

Control: 10 μl of a standard PLA₂ solution was used at a concentrationof 120 ng/10 μl in 20 mM Tris pH 8. Serial dilution was done by adding20 mM Tris pH 8 buffer to the final volume of 20 μl.

Inhibitor solution: Pentapeptide was usually dissolved in 1 μl of 0.1%TFA solution and further 9 μl of 20 mM Tris pH8 was added. This solutionwas always maintained around pH7-8. 10 μl of this inhibitor solution wasadded into 10 μl of PLA₂ solution. Incubation: all samples wereincubated at 37° C. for one hour.

PLA₂ solution: A standard PLA 2 solution was prepared in 20 mM TrispH8.0 so that 10 μl will give 50% (approx) hydrolysis.

Pentapeptide solution: A standard pentapeptide solution was made to 10mg/ml in 0.1% TFA. 100 μl was taken out and neutralised with 900 μl 20mM Tris pH8. 10 μl (10 μg was taken out for dose response together with10 μl of the PLA₂ solution). Serial dilution was carried out on 10 μlaliquots with 20 mM Tris pH 8.

Septic shock experiments:

Septic shock serum was diluted 1/100 for dose response experiments andused neat for serial dilution. Final reaction volume was always in theratio of 10 μl serum/10 μl Tris or pentapeptide solution.

Activity assay:

PLA₂ activity was measured using a mixed micellephosphatidylethanolamine (PE)/sodium deoxycholate assay, modified from amethod described by Seilhamer et al (1). The PE substrate was preparedby dissolving freshly desiccated PE (Amersham, Bucks, England) in 2%DOC, then diluting this to 0.22 nmoles PE and 0.04% DOC per sample inassay buffer (50 mM Tris-HCl, pH 8.5, 2 mM calcium chloride, 150 mMsodium chloride, 0.04% DOC). The sample was prepared by mixing 10 μl ofthe test material with 10 μl mM Tris-HCl pH7.4 and leaving at 37° C. for10 minutes. The reaction was started by the addition of 25 μl prewarmedsubstrate and terminated by addition of 10 μl 100 mM EDTA. The reactionmixture (30 μl was spotted and dried on silica TLC plates (Merck,Darmstadt, West Germany), and the plates were chromatographed usingchloroform:methanol:acetic acid (90:10:1) as solvent. The dried plateswere exposed overnight with Kodak X OMAT AR film. Radioactivity at theorigin and of the liberated arachidonic acid was counted and the percenthydrolysis by PLA 2 determined.

A summary of the results obtained with peptides corresponding toresidues 70-7u of several Type I and Type II enymes are set out inTable 1. These results show that there is considerable speciesspecificity in that peptides active against one species of PLA₂ were notactive against the other species tested. In addition none of thepeptides tested were active against PLA₂ type 1. This result indicatesthat inhibition by peptides from this region of PLA₂ (70-74) appears tooccur only on type II enymes.

Peptide 5 was shown to be an active inhibitor of human Type II PLA₂,however peptides 7, 8, 9, 10, 11 and 12 were all formed to be negative.This suggests that the peptide must be of a certain size to showinhibition and that inhibition will occur only with the specificsequence desired from the specific Type II enyme being tested.

                  TABLE 1                                                         ______________________________________                                        Type   II       II       II       I      I                                    Enzyme Syno     Crot.Dur.                                                                              Crot.Atr.                                                                              N.N.Atra                                                                             Por.Pan                              Inhibitor                                                                            PLA.sub.2                                                                              PLA.sub.2                                                                              PLA.sub.2                                                                              PLA.sub.2                                                                            PLA.sub.2                            ______________________________________                                        sPLA.sub.2                                                                           +        -        -        -      -                                    (FLSYK)                                                                       Crot.Dur                                                                             -        +        -        -      -                                    (WDIYR)                                                                       Crot.Atr                                                                             -        -        +        -      -                                    (TVSYT)                                                                       N.N.At -        -        -        -      -                                    (FKTYS)                                                                       Por.Pan                                                                              -        -        -        -      -                                    (TESYS)                                                                       ______________________________________                                         sPLA.sub.2 Human Type II PLA.sub.2                                            Crot.Dur  Crotalus decrissurs PLA.sub.2                                       Crot.Atr  Crotalus atrox PLA.sub.2                                            N.N.AT  Naja naja atrox PLA.sub.2                                             Por.Pan.  Porcine pancreatic PLA.sub.2                                   

From the above results the present inventors believe that short peptidesfrom the 70-74th region will most likely compete with the substrate foraccess to the active site and give inhibitory effects. It is believedthat variation of the length of the peptides present in these regionsmay result in a optimisation of the inhibition.

The pentapeptide apparently possesses helical structure (approximatelyone and a half turns). Since the helical structures are stablised byhydrogen bonds between the C═O of one residue and NH of the fourthresidue along the chain, the structure of the pentapeptide may besomewhat unstable and be more sensitive to the environment than a longerhelical molecule. On the other hand, it would be expected that the rangeof sizes that is effective will be limited because of the limited accessto the active site of PLA₂.

It is believed that the usual interchange of a hydrophobic residue e.g.Leu to Ile, Ser to Thr could also maintain the inhibitory effect. Thatis, amino acid residues alike in either charge or hydrophobicity couldpossibly be interchanged with those in the models without destroying thespecific interaction of the two regions. Since helix-helix interactionsare possibly the cause of the inhibitory action, small increases in thelength of the peptides could stablise this structure.

The results obtained in these studies also suggest that monoclonalantibodies could be raised against epitopes containing one or both ofthe peptide regions to effect a similar result on the PLA₂ activity.Such monoclonal antibodies could be produced using standard techniqueswell known in the art.

As will be apparent to those skilled in the art the principle of thepresent invention will also have application for the inhibition of theactivity of enzymes other than PLA₂ eg. the neuraminadase enzyme of theinfluenza virus or neuropeptide Y. It is envisaged that as biologicalactive proteins in general, possess an active conformation which isstabilized by interaction with the surrounding chain of amino acids,that peptides adjacent to, and capable of interaction with the residueswithin the active site will inhibit the activity of the enzyme. It isintended that such other peptides are included within the scope of thepresent invention.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

1. Johnson L. K. et al, Advance in Exp. Med & Biol; PLA 2 Role andFunction in Inflammation, P. Y-K Wong ed, PLenum Press 17-34 (1991).

2. Scott D. L. et al, Science 250, 1563 (1990).

3. Seilhamer J. J. et al;, J. Biol Chem 264, 5335 (1989).

4. Renetseder R. et al, J. Biol Chem 260, 11627 (1985)

5. Scott D. L. et al, Science 250, 1541 (1990).

6. White S. P. et al, Science 250, 1560 (1990).

7. Prozanski W. et al, J. Rheumatol., 15:1351-1355 (1988)

8. Vadas P., J. Lab. Clin. Med., 104:873-881 (1984)

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaLeuTrpGlnPheArgSerMetIleLysCysAlaIleProGlySer                              151015                                                                        HisProLeuMetAspPheAsnAsnTyrGlyCysTyrCysGlyLeuGly                              202530                                                                        GlySerGlyThrProValAspGluLeuAspArgCysCysGluThrHis                              354045                                                                        AspAsnCysTyrArgAspAlaLysAsnLeuAspSerCysLysPheLeu                              505560                                                                        ValAspAsnProTyrThrGluSerTyrSerTyrSerCysSerAsnThr                              65707580                                                                      GluIleThrCysAsnSerLysAsnAsnAlaCysGluAlaPheIleCys                              859095                                                                        AsnCysAspArgAsnAlaAlaIleCysPheSerLysAlaProTyrAsn                              100105110                                                                     LysGluHisLysAsnLeuAspThrLysLysTyrCys                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaValTrpGlnPheArgAsnMetIleLysCysThrIleProGlySer                              151015                                                                        AspProPheArgGluTyrAsnAsnTyrGlyCysTyrCysGlyLeuGly                              202530                                                                        GlySerGlyThrProValAspAspLeuAspArgCysCysGlnThrHis                              354045                                                                        AspHisCysTyrAsnGlnAlaLysLysLeuGluSerCysLysPheLeu                              505560                                                                        IleAspAsnProTyrThrAsnThrTyrSerTyrLysCysSerGlyAsn                              65707580                                                                      ValIleThrCysSerAspLysAsnAsnAspCysGluSerPheIleCys                              859095                                                                        AsnCysAspArgGlnAlaAlaIleCysPheSerLysValProTyrAsn                              100105110                                                                     LysGluTyrLysAspLeuAspThrLysLysHisCys                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 126 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaValTrpGlnPheArgLysMetIleLysCysValIleProGlySer                              151015                                                                        AspProPheLeuGluTyrAsnAsnTyrGlyCysTyrCysGlyLeuGly                              202530                                                                        GlySerGlyThrProValAspGluLeuAspLysCysCysGlnThrHis                              354045                                                                        AspAsnCysTyrAspGlnAlaLysLysLeuAspSerCysLysPheLeu                              505560                                                                        LeuAspAsnProTyrThrHisThrTyrSerTyrSerCysSerGlySer                              65707580                                                                      AlaIleThrCysSerSerLysAsnLysGluCysGluAlaPheIleCys                              859095                                                                        AsnCysAspArgAsnAlaAlaIleCysPheSerLysAlaProTyrAsn                              100105110                                                                     LysAlaHisLysAsnLeuAspThrLysLysTyrCysGlnSer                                    115120125                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AsnLeuValAsnPheHisArgMetIleLysLeuThrThrGlyLysGlu                              151015                                                                        AlaAlaLeuSerTyrGlyPheTyrGlyCysHisCysGlyValGlyGly                              202530                                                                        ArgGlySerProLysAspAlaThrAspArgCysCysValThrHisAsp                              354045                                                                        CysCysTyrLysArgLeuGluLysArgGlyCysGlyThrLysPheLeu                              505560                                                                        SerTyrLysPheSerAsnSerGlySerArgIleThrCysAlaLysGln                              65707580                                                                      AspSerCysArgSerGlnLeuCysGluCysAspLysAlaAlaAlaThr                              859095                                                                        CysPheAlaArgAsnLysThrThrTyrAsnLysLysTyrGlnTyrTyr                              100105110                                                                     SerAsnLysHisCysArgGlySerThrProArgCys                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 125 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       SerLeuLeuGluPheGlyGlnMetIleLeuPheLysThrGlyLysArg                              151015                                                                        AlaAspValSerTyrGlyPheTyrGlyCysHisCysGlyValGlyGly                              202530                                                                        ArgGlySerProLysAspAlaThrAspGluCysCysValThrHisGlu                              354045                                                                        CysCysTyrAsnArgLeuGluLysSerGlyCysGlyThrLysPheLeu                              505560                                                                        ThrTyrLysPheSerTyrArgGlyGlyGlnIleSerCysSerThrAsn                              65707580                                                                      GlnAspSerCysArgLysGlnLeuCysGlnCysAspLysAlaAlaAla                              859095                                                                        GluCysPheSerArgAsnLysLysSerTyrSerLeuLysTyrGlnPhe                              100105110                                                                     TyrProAsnLysPheCysLysXaaXaaThrProSerCys                                       115120125                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AspLeuLeuAsnPheArgLysMetIleLysLeuLysThrGlyLysAla                              151015                                                                        ProValProAsnTyrAlaPheTyrGlyCysTyrCysGlyLeuGlyGly                              202530                                                                        LysGlySerProLysAspAlaThrAspXaaCysCysAlaAlaHis                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       HisLeuLeuAspPheArgLysMetIleArgTyrThrThrGlyLysGlu                              151015                                                                        AlaThrThrSerTyrGlyAlaTyrGlyCysHisCysGlyValGlyGly                              202530                                                                        ArgGlyAlaProLysXaaAlaLysPheLeuSerTyrLysPheSerMet                              354045                                                                        LysLysAlaAlaAlaAlaCysPheGlnPheTyrProAlaAsnArgCys                              505560                                                                        SerGlyArgProProSerCys                                                         6570                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       PheLeuSerTyrLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       LysPheLeuSerTyr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TrpAspIleTyrArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ThrValSerTyrThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ThrValSerThrThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      PheLysThrTyrSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ThrGluSerTyrSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyThrLysPheLeuSerTyrLysPheSerAsn                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      LysPheLeuSerTyrTyr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      PheLeuSerTyr                                                                  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We claim:
 1. A linear or cyclic peptide of at least 5 residues whichinhibits the enzymatic activity of human synovial PLA₂, the peptidehaving the following formula:

    A.sub.1 -A.sub.2 -A.sub.3 -A.sub.4 -A.sub.5 -A.sub.6

in which A₁ is K or R or H or absent A₂ is F or Y or W A₃ is L or V or Ior M A₄ is S or T A₅ is Y or F or W A₆ is K or R or H or absent.
 2. Apeptide as claimed in claim 1 in which the peptide is FLSYK or KFLSY. 3.A peptide as claimed in claim 1 in which the phospholipase A₂ is humanphospholipase A₂.
 4. A composition for use in treating the subjectsuffering from rheumatoid arthritis, septic shock and/or inflammatorydisease, the composition comprising a therapeutically effective amountof the peptide as claimed in claim 1 and a pharmaceutically acceptablesterile carrier.
 5. A peptide as claimed in claim 1, in which either A₁or A₆ is absent.
 6. A linear peptide which inhibits the enzymaticactivity of Crotalus durissus PLA₂, the peptide having the followingformula:

    B.sub.2 -B.sub.3 -B.sub.4 -B.sub.5 -B.sub.6

in which B₂ is W or F or Y B₃ is D or E B₄ is I or V or L or M B₅ is Yor F or W B₆ is R or K or H.
 7. A peptide as claimed in claim 6 in whichthe peptide is WDIYR.
 8. A linear peptide which inhibits the enzymaticactivity of Crotalus atrox PLA₂, the peptide having the followingformula:

    C.sub.2 -C.sub.3 -C.sub.4 -C.sub.5 -C.sub.6

in which C₂ is T or S C₃ is V or I or L or M C₄ is T or S C₅ is Y or For W C₆ is T or S.
 9. A peptide as claimed in claim 8 in which thepeptide is TVSYT.